PROJECT SUMMARY A justifiable lack of access to human embryos for experimental purposes has presented a longstanding barrier to the in vitro investigation of human organogenesis. To circumvent this impasse, the overall goal of this R21 proposal is to establish a powerful new 3-D experimental model system that recapitulates key structural and biophysical elements of the embryonic niche environment to enable investigation of human cardiac morphogenesis and development. We propose a synergistic combination of innovative bioreactor design, state-of-the-art human pluripotent stem cell biology, and cutting-edge human cardiac tissue engineering technology, with a quantitative approach based on sound bioengineering principles. The strategy is to develop a microfluidics based bioreactor system to grow endocardial tubes, myocardium, and cardiac jelly using human pluripotent stem cell (hPSC) derived cardiomyocytes, fibroblasts and endocardial cells in order to study the early developmental stages of heart tube formation in vitro. By providing fluid shear forces, mechanical constraints, and morphogen gradients similar to those experienced by the developing heart tube, and by seeding the bioreactor with spatially compartmentalized cell types in starting positions that mimic the primitive anatomy, we aim to provide a niche environment in which they can begin building a heart as they would in nature. The two specific aims of this proposal are consistent with the R21 Exploratory/Developmental Bioengineering Research Grant mechanism (PA-16-040): Aim 1 is to develop a microfluidic bioreactor with boundary constraints and flow control for the creation and evaluation of engineered morphogenetic human heart organoids. Aim 2 is to establish an in vitro model of human congenital heart tube malformation, exploring chemical and genetic interventions known to cause heterotaxy in vivo. This exploratory and developmental proposal offers a desperately needed and potentially disruptive leap forward in the sophistication of available in vitro human model systems. The multidisciplinary expertise of the research team, combined with the available state-of-the-art resources and facilities, provides a unique opportunity to overcome the anticipated technical challenges and successfully achieve the proposed aims. This is therefore considered a high-risk, high-impact proposal, likely to yield new tools and insights for understanding the process of cardiac tube formation, for creating more realistic multi-tissue heart organoids, and for eventually studying diseases related to cardiac structural defects using a unique anatomical in vitro 3-D human heart model system.